Fecal shedding of Escherichia coli O157:H7 in cattle, except those that shed transiently, is due to the organism's ability to persist in the gut. Site of prevalence in the gut is important for understanding the mechanisms and factors affecting gut persistence and fecal shedding and is a potential target for intervention. The prevalence of E. coli O157:H7 in the rumen, cecum, colon, and rectum was determined with contents collected from slaughtered cattle (n = 815) at an abattoir. Isolation and identification of E. coli O157:H7 were by selective enrichment, immunomagnetic separation, plating on selective medium, agglutination for O157 antigen, and presence of virulence genes. Prevalence in the rumen, cecum, colon, and rectum was 4.9%, 9.9%, 7.6%, and 11.1%, respectively. The overall prevalence of E. coli O157:H7 in the cattle sampled, based on being positive in any one gut location, was 20.3%. E. coli O157:H7 in rectal contents was positively associated (p < 0.01) with presence in the rumen or colon but not in the cecum. Pulsed-field gel electrophoresis (PFGE) was performed to compare the clonal similarity of isolates (n = 144) obtained from the rectum with that of rumen, cecum, or colon within cattle (n = 77). The majority (79-90%) of isolates obtained within the same animal shared a common PFGE type. There were no significant differences in PFGE type between positive samples from the rectum and samples from other locations within the same animal. Acid tolerance for cattle with positive rumen (pregastric) isolates and with at least one other positive hindgut (postgastric) isolate within the same animal was determined. There was no significant difference between gut locations in log reduction following acid challenge. The hindgut was the major site of prevalence of E. coli O157:H7 in cattle, a majority of the isolates within the same animal were clonally similar, and acid tolerance of hindgut isolates were not different from that of ruminal isolates.